JP6574414B2 - Compositions, methods and devices for dialysis - Google Patents

Description

  The section headings used herein are for organizational purposes only and do not limit the contents described.

  This application claims priority to US Provisional Application No. 61 / 832,235, filed June 7, 2013, the entire contents of which are incorporated by reference.

Oxalic acid is a dicarboxylic acid of the formula _{HO 2} C-CO 2 H. Oxalic acid is present mainly in biological organisms as oxalate and is a salt form of oxalic acid. Oxalates are found in foods such as spinach, dairy, strawberry, cranberry, nuts, cocoa, chocolate, peanut butter, sorghum, and tea. Oxalates are also metabolic end products of humans and other mammals. Oxalate is excreted in the urine by the kidneys. Oxalic acid, when mixed with calcium, produces calcium oxalate, an insoluble product, the most common compound found in kidney stones.

  Because mammals do not synthesize enzymes that break down oxalates, an individual's oxalate concentration is usually regulated by filtration and excretion through the kidneys and a small uptake of about 5-10% of food oxalate through the gastrointestinal tract. High oxalate levels can cause a variety of medical conditions such as hyperoxaluria, oxalate, primary hyperoxaluria, intestinal hyperoxaluria, idiopathic hyperoxaluria, or end-stage renal disease (ESRD) Come. For example, Hoppe, Nat. Rev. Nephrol. 8: 467-75 (2012). The increase in oxalate can be caused by excessive intake of oxalate from food, excess absorption of oxalate from the intestine and abnormal production of endogenous oxalate. Intestines due to overabsorption of oxalate in the colon and small intestine, including bile acid and fat metabolism disorders, ileectomy, and overabsorption caused by, for example, celiac disease, exocrine pancreatic insufficiency, intestinal disease, and fatty diarrhea due to liver disease May cause illness.

  Hyperoxalicemia or elevated blood oxalate can be caused, for example, by decreased oxalate excretion in patients with renal failure. Hyperoxalate can cause precipitation of calcium oxalate in body tissues or organs and is called oxalate. Oxalosis often results in high oxalicuria, including calcium oxalate deposition in kidney tissue (renal calcification) or urinary tract (eg, kidney stones, urolithiasis, and kidney stones). Calcium oxalate can, for example, deposit on and damage the eyeballs, blood vessels, joints, bones, muscles, heart and other major organs. See, for example, Leumann et al. , J .; Am. Soc. Nephrol. 12: 1986 1993 (2001) and Monico et al. , Kidney International 62: 392 400 (2002). The effect of increasing oxalate concentration can appear in various tissues. For example, when it deposits on small blood vessels, it causes painful skin ulcers that do not heal, when it deposits on bone marrow, it causes anemia, when it deposits on bone tissue, fractures affect the growth of children, and when calcium oxalate deposits on the heart Causes abnormal cardiac rhythm or poor cardiac function. Hyperoxaluria often progresses to calcium oxalate stone formation and / or medullary nephrocalcinosis. As a result, this process resulted in a decrease in glomerular filtration rate, an increase in plasma oxalate concentration, and deposition of calcium oxalate crystals in parenchymal organs such as bones, joints, heart, and retina, resulting in systemic oxalate Collectively. Hoppe et al. , Am. J. et al. Nephrol. 25: 276-281 (2005). Decreasing plasma oxalate levels caused oxalate (tissue deposits of oxalate), nephrocalcinosis (calcium-oxalate interstitial deposits), and severe primary hyperoxaluria (PH) patients, and end-stage renal disease (ERSD) Deterioration of calcium oxalate kidney stones in patients can be prevented. Hoppe, Nat. Rev. Nephrol. 8: 467-75 (2012); Hoppe et al. , Kidney Int. 56: 268-74 (1999).

  Existing methods for treating elevated oxalate concentrations have not been shown to be effective, and concentrated dialysis and organ transplantation are often used in patients with primary hyperoxaluria, intestinal hyperoxaluria, or ESRD It seems necessary. Various existing treatments for hyperoxaluria include high doses of pyridoxine, orthophosphate, magnesium, iron, aluminum, potassium citrate, and cholestyramine, as well as diet and water intake adjustment, dialysis, and surgery Treatment, for example, for renal and liver transplantation. In existing peritoneal dialysis methods, removal of oxalate is not sufficient to remove excess endogenous oxalate. That is, the removal rate is lower than the generation rate. Marangella et al. , Contrib. Nephrol. 136: 11-32 (2001). For example, conventional peritoneal dialysis is not sufficient to remove a sufficient amount of oxalate, especially for patients with systemic oxalate who have experienced ESRD. Cochat et al. , Nephrol. Dial. Transplant. 27: 1279-36 (2012). For this reason, the body's oxalate accumulation rapidly increases even with concentrated dialysis. Patients also need constant monitoring to adjust dialysis therapy based on plasma oxalate concentration. In fact, the combination of hemodialysis and peritoneal dialysis does not reduce the body's oxalate concentration, and of course there is no form of dialysis that can accommodate the oxalate produced in the body of high oxalate. Understood. Hoppe et al. Nephrol Dial Transplant. 19: 39-42 (2004). For this reason, patients must tolerate concentrated hemodialysis with 5-6 sessions of 5 hours / week and even night peritoneal dialysis until a kidney transplant is performed. Hoppe et al. , (2004).

In patients who develop ESRD, treatment with vigorous (6 or 7 times per week) daily hemodialysis (with or without supplementary peritoneal dialysis) while the patient waits for kidney transplantation is a temporary treatment Only. However, because the liver is the main cause of excess oxalate production in individuals with primary hyperoxaluria, patients who have only undergone kidney transplantation eventually develop renal failure due to progression of oxalate deposits in the new kidney Is likely to recur. Only a combination of kidney and liver transplantation or preemptive liver transplantation has been described as a treatment for treating PH patients. Hoppe et al. , Pediatr. Nephrol. 18: 986-91 (2003). However, these methods are a significant risk to patients on their own and on their own. Until the identification of genetic treatments or treatments not involved in organ transplantation, the discovery of new therapies that help treat PH will greatly help limit morbidity and mortality in this patient population.
Thus, there is a need for treatment methods that more effectively reduce the concentration of oxalate or other metabolites in a patient. More specifically, there is an urgent need for a method of treating plasma oxalate or other metabolite accumulation that is less burdensome than hemodialysis and organ transplantation. There is a further need for in vitro and in vivo models to evaluate the effectiveness and regulatory parameters of such peritoneal dialysis methods.

Hoppe, Nat. Rev. Nephrol. 8: 467-75 (2012) Leumann et al. , J .; Am. Soc. Nephrol. 12: 1986 1993 (2001) Monico et al. , Kidney International 62: 392 400 (2002). Hoppe et al. , Am. J. et al. Nephrol. 25: 276-281 (2005) Hoppe et al. , Kidney Int. 56: 268-74 (1999) Marangella et al. , Contrib. Nephrol. 136: 11-32 (2001) Cochat et al. , Nephrol. Dial. Transplant. 27: 1279-36 (2012) Hoppe et al. Nephrol Dial Transplant. 19: 39-42 (2004) Hoppe et al. , Pediatr. Nephrol. 18: 986-91 (2003)

  This publication describes the composition of peritoneal dialysis solutions and metabolic enzymes to treat disorders that are pathologically associated with increased concentrations of metabolites, including, for example, oxalate-related disorders such as oxalic acid and hyperoxalicemia. Things and their use. In some embodiments, for example, a peritoneal dialysis solution supplemented with metabolic enzymes by peritoneal dialysis can be administered to a mammal to effectively reduce the oxalate concentration in the blood circulation of the mammal, and / or oxalate Damage due to deposits can be reduced. The compositions and methods of the present invention can be implemented easily and conveniently.

  Peritoneal dialysis solutions or functional fragments thereof with the addition of metabolic enzymes can improve the removal of harmful metabolites from the plasma of patients undergoing dialysis. For example, peritoneal dialysis solutions supplemented with oxalate degrading enzymes can significantly improve the effectiveness of peritoneal dialysis in patients with oxalate and kidney failure. The present invention allows for the rapid degradation of metabolites, such as oxalate, allowing removal of the peritoneal cavity from the peritoneal dialysis solution into the peritoneal dialysis solution between the patient's circulation and peritoneal dialysis solution over time. Maintain or increase the metabolite gradient. Such a gradient further improves the removal of unwanted metabolites from the circulation, significantly reduces plasma metabolite concentrations, and consequently reduces the concentration of metabolites in tissues such as the eye, kidney or liver. Let When a peritoneal dialysis solution is administered to a patient's peritoneal cavity, metabolic enzymes in the solution degrade harmful metabolites, such as oxalate, in the peritoneal cavity. Thus, the peritoneal dialysis compositions and methods described herein can provide significant improvements over existing dialysis methods. The compositions and methods described herein can remove harmful metabolites from the bloodstream more quickly and effectively than existing peritoneal dialysis methods, resulting in shorter peritoneal dialysis sessions given to patients. And / or less. In addition, in the combined treatment of hemodialysis and peritoneal dialysis, the compositions and methods described herein can improve the effectiveness of peritoneal dialysis, resulting in the number and duration of required hemodialysis sessions. Thus reducing the physical and economic burden on patients undergoing dialysis treatment. Peritoneal dialysis using the compositions described herein also exhibits significant advantages over other extracorporeal circulation devices that use metabolic enzymes to filter body fluids outside the body. Compared to metabolic enzymes that are limited to extracorporeal circulation devices, the metabolic enzymes are administered using the compositions and methods of the present invention, creating a gradient of harmful metabolites between the various compartments of the body, Can be promoted and increased.

  In a first aspect, the present disclosure provides a composition comprising a peritoneal dialysis solution and at least one metabolic enzyme or functional fragment thereof. In some embodiments, the metabolic enzyme is, for example, an oxalate degrading enzyme (eg, oxalate oxidase, oxalate decarboxylase, oxalyl-CoA decarboxylase, and formyl CoA transferase), a urate degrading enzyme (eg, urate oxidase (uricase)). )), Ureolytic enzymes (eg urease), bilirubin degrading enzymes (eg bilirubin oxidase), and phenylalanine degrading enzymes (eg phenylalanine hydroxylase, phenylalanine ammonia lyase). In some embodiments, the metabolic enzyme is oxalate decarboxylase. In some embodiments, the metabolic enzyme (eg, oxalate degrading enzyme) can be a covalent modification, eg, pegylation. In some embodiments, the metabolic enzyme can be soluble or crystalline. In some embodiments, the metabolic enzyme can be cross-linked or non-cross-linked. In some embodiments, the metabolic enzyme can comprise a sequence that is identical or substantially identical to an enzyme sequence found in a natural source, eg, a plant, bacterium, or fungus. In some embodiments, metabolic enzymes can be produced by genetic recombination.

  In some embodiments, the peritoneal dialysis solution can include at least one osmotic agent, at least one electrolyte, and at least one organic acid salt. In certain embodiments, the compositions described herein reduce mammalian oxalate concentrations, eg, oxalate concentrations in bodily fluids selected from urine, blood, plasma, serum, and peritoneal fluid. it can.

  This publication further provides methods for reducing the concentration of mammalian metabolites, such as oxalate. In some embodiments, the method can include administering a peritoneal dialysis solution described herein to the mammal. In some embodiments, the method can include preparing a peritoneal dialysis solution comprising at least one metabolic enzyme and administering an enzyme-added peritoneal dialysis solution to the mammal. In some embodiments, the metabolic enzyme is, for example, an oxalate degrading enzyme (eg, oxalate oxidase, oxalate decarboxylase, oxalyl-CoA decarboxylase, and formyl CoA transferase), a urate degrading enzyme (eg, urate oxidase (uricase)). )), Ureolytic enzymes (eg, urease), bilirubin degrading enzymes (eg, bilirubin oxidase), and phenylalanine degrading enzymes (eg, phenylalanine hydroxylase, phenylalanine ammonia lyase), and the function of any one of these enzymes You can select from fragments. In some embodiments, the metabolite is selected from oxalate, uric acid, urea, bilirubin, and phenylalanine. In some embodiments, the metabolite is oxalate. In some embodiments, the metabolic enzyme is oxalate decarboxylase. Dialysis with a peritoneal dialysis solution containing one or more metabolic enzymes increases the effectiveness of oxalate removal and is associated with increased metabolite concentrations compared to existing dialysis methods involving extracorporeal circulation devices containing metabolic enzymes Can improve the treatment of medical conditions. In some embodiments, the method further comprises a mammalian biological sample, eg, a metabolite. Detecting the concentration of a metabolite, such as oxalate, in urine, blood, plasma, serum, or peritoneal fluid of a patient suspected of having a pathological increase in concentration. In some embodiments, peritoneal dialysis using the compositions and methods of the invention reduces the concentration of a metabolite, such as oxalate, by at least 10%, such as at least 20%, at least 30%, or at least 40% or more. be able to. Such a decrease in concentration can be measured, for example, in a biological sample selected from urine, blood, plasma, serum, and peritoneal fluid. In certain embodiments, the methods described herein can be combined with other methods of reducing mammalian metabolite concentrations, such as hemodialysis.

  In another aspect, the present disclosure provides a method of treating, preventing and / or delaying the progression of a disorder associated with increased mammalian metabolite concentration by administering to a mammal a peritoneal dialysis solution of the present invention. To do. In some embodiments, the method can include adding at least one metabolic enzyme to the peritoneal dialysis solution and administering the added peritoneal dialysis solution to the mammal. In some embodiments, the metabolic enzyme is, for example, an oxalate degrading enzyme (eg, oxalate oxidase, oxalate decarboxylase, oxalyl-CoA decarboxylase, and formyl CoA transferase), a urate degrading enzyme (eg, urate oxidase (uricase)). )), Ureolytic enzymes (eg urease), bilirubin degrading enzymes (eg bilirubin oxidase), and phenylalanine degrading enzymes (eg phenylalanine hydroxylase, phenylalanine ammonia lyase). In some embodiments, the metabolic enzyme is oxalate decarboxylase. In some embodiments, the metabolite is selected from oxalate, uric acid, urea, bilirubin, and phenylalanine. In some embodiments, the metabolite is oxalate. In some embodiments, the disorder is high secondary to oxalic acid disease, hyperoxalicemia, and a disease selected from kidney disorder, bone disorder, liver disorder, gastrointestinal disorder, and pancreatic disorder. It can be selected from oxalicemia. For example, disorders include oxalate, hyperoxalate, primary hyperoxaluria, intestinal hyperoxaluria, idiopathic hyperoxaluria, and end-stage renal disorder, ethylene glycol addiction, cystic pulmonary fibrosis, One can select from secondary hyperoxalates for diseases selected from inflammatory bowel disease, urolithiasis, nephrolithia, and chronic kidney disease. In some embodiments, the disorder is uric acid, urea, bilirubin, or phenylalanine, such as, for example, hyperuricemia, gout, uremia, hyperbilirubinemia, jaundice, hyperphenylalaninemia, and phenorketonuria (PKU). ) Is associated with an increase in concentration. In certain embodiments, the methods described herein can be combined with other treatment methods for increasing metabolite concentrations, such as hemodialysis.

In addition, an animal model of hyperoxalicemia is provided. The animal model described herein appears to be similar to the condition of patients with primary hyperoxaluria (PH) disease and / or end stage renal disease (ESRD). In some embodiments, the model can include a non-human mammal, such as a pig, to which oxalate is administered by continuous infusion. At the functional level, human and porcine species have many similarities in gastrointestinal and genitourinary structures. Unlike rodents, humans and pigs have polypyramidal kidneys with similar characteristics in maximum urinary concentration, glomerular filtration rate, and total renal blood flow. Mendel et al. , J Urol. 171: 1301-03 (2004); Kaplon et al. , J .; Endourol. 24: 355-59 (2010). A first device can be inserted into the mammal to administer oxalate with continuous infusion. In some embodiments, the first instrument can be, for example, a catheter or a pleural trocar. The catheter can be inserted percutaneously with or without surgery. In some embodiments, the catheter can be a peripheral venous catheter, such as VENFLON ™ or CATHLON ™. In some embodiments, a second device, such as a catheter, can be inserted into the mammal for blood collection. The methods described herein can be performed using animal models. As shown in the Examples, peritoneal dialysis (PD) performed in one embodiment of the animal model described herein typically allowed blood to flow during approximately 4-6 hours of treatment, which is usually one cycle of PD. About 20% to 30% of the soy oxalate can be removed.
In the embodiment of the present invention, for example, the following items are provided.
(Item 1)
A composition comprising a peritoneal dialysis solution and at least one metabolic enzyme or functional fragment thereof.
(Item 2)
The composition according to item 1, wherein the at least one metabolic enzyme is selected from oxalate degrading enzyme, uric acid degrading enzyme, urea degrading enzyme, bilirubin degrading enzyme, and phenylalanine degrading enzyme.
(Item 3)
Item 3. The composition according to Item 2, wherein the oxalate degrading enzyme is selected from oxalate oxidase, oxalate decarboxylase, oxalyl-CoA decarboxylase, and formyl CoA transferase.
(Item 4)
Item 4. The composition according to Item 3, wherein the oxalate degrading enzyme is oxalate decarboxylase.
(Item 5)
Item 3. The composition according to Item 2, wherein the urate degrading enzyme is urate oxidase (uricase).
(Item 6)
Item 3. The composition according to Item 2, wherein the ureolytic enzyme is urease.
(Item 7)
Item 3. The composition according to Item 2, wherein the bilirubin degrading enzyme is bilirubin oxidase.
(Item 8)
Item 3. The composition according to Item 2, wherein the phenylalanine degrading enzyme is phenylalanine hydroxylase or phenylalanine ammonia lyase.
(Item 9)
Item 9. The composition according to any one of Items 1 to 8, wherein the at least one metabolic enzyme is covalently modified.
(Item 10)
10. A composition according to item 9, wherein the at least one metabolic enzyme is PEGylated.
(Item 11)
The composition according to any of items 1 to 10, wherein the at least one metabolic enzyme is soluble or crystalline.
(Item 12)
11. A composition according to any of items 1 to 10, wherein the at least one metabolic enzyme is crosslinked or not crosslinked.
(Item 13)
13. A composition according to any of items 1 to 12, wherein the peritoneal dialysis solution comprises at least one osmotic agent, at least one electrolyte, and at least one organic acid salt.
(Item 14)
14. A composition according to any of items 1 to 13, wherein the composition reduces a mammalian metabolite concentration.
(Item 15)
15. A composition according to item 14, wherein the metabolite is selected from oxalate, uric acid, urea, bilirubin, and phenylalanine.
(Item 16)
The composition is selected from the contents of urine, blood, plasma, serum, peritoneal fluid, eyeball, bone, kidney, liver, heart, and stomach, proximal small intestine, distal small intestine, cecum, colon, and rectum 16. The composition according to item 14 or 15, which reduces the metabolite concentration of a biological sample.
(Item 17)
A method of reducing a mammal metabolite concentration comprising administering to a mammal an added peritoneal dialysis solution comprising at least one metabolic enzyme or functional fragment thereof.
(Item 18)
18. The method of item 17, wherein the method comprises adding the at least one metabolic enzyme or functional fragment thereof to a peritoneal dialysis solution and administering the added peritoneal dialysis solution to the mammal.
(Item 19)
Item 19. The method according to item 17 or 18, wherein the at least one metabolic enzyme is selected from oxalate degrading enzyme, urate degrading enzyme, urea degrading enzyme, bilirubin degrading enzyme, and phenylalanine degrading enzyme.
(Item 20)
20. A method according to item 19, wherein the oxalate degrading enzyme is selected from oxalate oxidase, oxalate decarboxylase, oxalyl-CoA decarboxylase, and formyl CoA transferase.
(Item 21)
Item 21. The method according to Item 20, wherein the oxalate degrading enzyme is oxalate decarboxylase.
(Item 22)
Item 20. The method according to Item 19, wherein the urate decomposing enzyme is urate oxidase (uricase).
(Item 23)
Item 20. The method according to Item 19, wherein the ureolytic enzyme is urease.
(Item 24)
Item 20. The method according to Item 19, wherein the bilirubin degrading enzyme is bilirubin oxidase.
(Item 25)
Item 20. The method according to Item 19, wherein the phenylalanine degrading enzyme is phenylalanine hydroxylase or phenylalanine ammonia lyase.
(Item 26)
26. A method according to any of items 19 to 25, wherein the at least one metabolic enzyme is covalently modified.
(Item 27)
27. The method of item 26, wherein the at least one metabolic enzyme is PEGylated.
(Item 28)
28. A method according to any of items 19 to 27, wherein the at least one metabolic enzyme is soluble or crystalline.
(Item 29)
29. A method according to any of items 19 to 28, wherein the at least one metabolic enzyme is cross-linked or not cross-linked.
(Item 30)
30. A method according to any of items 19 to 29, wherein the peritoneal dialysis solution comprises at least one osmotic agent, at least one electrolyte, and at least one organic acid salt.
(Item 31)
31. A method according to any of items 19 to 30, wherein the metabolite is selected from oxalate, uric acid, urea, bilirubin, and phenylalanine.
(Item 32)
32. The method according to any of items 19 to 31, further comprising detecting a metabolite concentration in the mammalian biological sample.
(Item 33)
33. The method of item 32, wherein the biological sample is selected from urine, blood, plasma, serum, and peritoneal fluid, or taken from tissue.
(Item 34)
34. A method according to any of items 19 to 33, wherein said administration reduces the metabolite concentration by at least 10%.
(Item 35)
The decrease is selected from the contents of urine, blood, plasma, serum, and peritoneal fluid, eyeball, bone bone, kidney, liver, heart, and stomach, proximal small intestine, distal small intestine, cecum, colon, and rectum 35. The composition according to item 34, which is measured with a biological sample to be measured.
(Item 36)
36. A method according to any of items 19 to 35, wherein the method is combined with hemodialysis.
(Item 37)
A method of treating a disorder associated with increased mammalian metabolite concentration, comprising administering an additional peritoneal dialysis solution comprising at least one metabolic enzyme or functional fragment thereof to said mammal in a therapeutically effective amount. Including said method.
(Item 38)
39. The method of item 38, wherein the method comprises adding the at least one metabolic enzyme or functional fragment thereof to a peritoneal dialysis solution and administering the added peritoneal dialysis solution to the mammal.
(Item 39)
39. A method according to item 37 or 38, wherein the at least one metabolic enzyme is selected from oxalate degrading enzyme, urate degrading enzyme, urea degrading enzyme, bilirubin degrading enzyme, and phenylalanine degrading enzyme.
(Item 40)
40. The method of item 39, wherein the oxalate degrading enzyme is selected from oxalate oxidase, oxalate decarboxylase, oxalyl-CoA decarboxylase, and formyl CoA transferase.
(Item 41)
41. A method according to item 40, wherein the oxalate degrading enzyme is oxalate decarboxylase.
(Item 42)
40. The method of item 39, wherein the urate degrading enzyme is urate oxidase (uricase).
(Item 43)
40. The method of item 39, wherein the ureolytic enzyme is urease.
(Item 44)
40. A method according to item 39, wherein the bilirubin degrading enzyme is bilirubin oxidase.
(Item 45)
40. The method of item 39, wherein the phenylalanine degrading enzyme is phenylalanine hydroxylase or phenylalanine ammonia lyase.
(Item 46)
46. A method according to any of items 39 to 45, wherein the at least one metabolic enzyme is covalently modified.
(Item 47)
47. The method of item 46, wherein the at least one metabolic enzyme is PEGylated.
(Item 48)
48. A method according to any of items 39 to 47, wherein the at least one metabolic enzyme is soluble or crystalline.
(Item 49)
49. A method according to any of items 39 to 48, wherein the at least one metabolic enzyme is cross-linked or not cross-linked.
(Item 50)
50. A method according to any of items 39 to 49, wherein the peritoneal dialysis solution comprises at least one osmotic agent, at least one electrolyte, and at least one organic acid salt.
(Item 51)
51. A method according to any of items 39 to 50, wherein the metabolite is selected from oxalate, uric acid, urea, bilirubin, and phenylalanine.
(Item 52)
The disorder is oxalate, hyperoxalate, or hyperoxalate secondary to a disease selected from kidney disorder, bone disorder, liver disorder, gastrointestinal disorder, and pancreatic disorder, 41. A method according to item 39 or 40.
(Item 53)
The disorders are oxalic acid, hyperoxalicemia, primary hyperoxaluria, intestinal hyperoxaluria, idiopathic hyperoxaluria, and end-stage renal disorder, ethylene glycol poisoning, cystic pulmonary fibrosis, inflammatory 55. The method of item 54, wherein the oxalate is secondary to a disease selected from bowel disease, urolithiasis, nephrolithia, and chronic kidney disease.
(Item 54)
41. The method of item 39 or 40, wherein the disorder is selected from hyperuricemia, gout, uremia, hyperbilirubinemia, jaundice, hyperphenylalaninemia, and phenorketonuria (PKU).
(Item 55)
55. A method according to any of items 39 to 54, wherein the method is combined with hemodialysis.
(Item 56)
A porcine model of hyperoxalicemia, including pigs administered with continuous infusion of oxalate.
(Item 57)
59. A pig model according to item 56, wherein a first device is inserted into the pig for administering oxalate in a continuous infusion.
(Item 58)
58. A pig model according to item 57, wherein the first device is a catheter.
(Item 59)
59. A pig model according to item 57 or 58, wherein a second instrument is inserted into the pig for blood collection.
(Item 60)
A method of generating a porcine model of hyperoxalate, comprising administering oxalate to a pig by continuous infusion.
(Item 61)
60. introducing an additional peritoneal dialysis solution comprising at least one metabolic enzyme or a functional fragment thereof into the peritoneal cavity, and administering the added peritoneal dialysis solution to a pig model according to any of items 56 to 59 A method of peritoneal dialysis comprising:

The details of one or more embodiments of the disclosure are set forth in the accompanying drawings and the description below.
Oxalate concentrations in serum and peritoneal dialysate (PD) samples from pigs infused with various concentrations of oxalate are shown (FIGS. 1a and 1b). Oxalate concentrations in serum and peritoneal dialysate (PD) samples from pigs infused with various concentrations of oxalate are shown (FIGS. 1a and 1b). The oxalate concentration is shown in serum and peritoneal dialysate samples from pigs infused with oxalate and dosed with PD (control) or PD + OXDC. 1 shows a diagram of an in vitro dialysis model using PD solution GAMBROSOL TRIO ™ 10 ± OXDC. Figure 5 shows oxalate concentration in an in vitro dialysis model using GAMBROSOL TRIO ™ 10 ± OXDC. FIG. 2 shows a diagram of an in vitro dialysis model using serum and PD solution GAMBROSOL TRIO ™ 10 ± OXDC. The oxalate concentration of the in vitro dialysis model using serum and GAMBROSOL TRIO ™ 10 ± OXDC is shown. 2 shows an OXDC dose-dependent decrease in oxalate concentration in in vitro serum.

  This publication is based in part on the composition of peritoneal dialysis solution and metabolic enzymes. Also described herein are methods of administering the compositions to reduce the concentration of metabolites and treat conditions associated with increased concentrations of one or more metabolites, such as oxalate-related disorders. Yes. In addition, animal models of hyperoxalicemia and peritoneal dialysis are described herein.

Definitions To make it easier to understand this publication, certain terms are first defined. These definitions should be read in light of the remainder of this disclosure as understood by those skilled in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Additional definitions are provided throughout the detailed description.

  The articles “a” and “an” as used herein should be understood to mean “at least one” unless clearly stated to the contrary.

  The phrase “and / or” as used herein means “one or both” of an element and the elements are combined, ie, in some cases the elements are combined and other In some cases it must be understood that they exist disjunctive. Unless explicitly stated to the contrary, there may optionally be other elements other than those explicitly specified by the “and / or” section, whether or not explicitly related to the specifically specified element. be able to. As a result, as a non-limiting example, referring to “A and / or B” when used in connection with open-ended terms, eg, “includes”, in one embodiment, A without A (optionally In another embodiment, B without A (optionally including elements other than A), and in yet another embodiment, both A and B (optionally including other elements) Including).

  In general, the term “or” as used herein is, for example, an exclusivity of “any”, “one of them”, “only one” or “exactly one”. Should be interpreted as indicating only an exclusive alternative (ie, “one or the other, not both”).

  “Consisting essentially of”, when used in the claims, shall have its ordinary meaning as used in the field of patent law.

  As used herein, the phrase “at least one” associated with one or more element lists means at least one element selected from one or more arbitrary elements of the element list, but within the element list. It should be understood that at least one and each of the elements explicitly listed in the table need not necessarily be included, and does not exclude any combination of elements in the element list. In this definition, there may be any element other than a clearly identified element in the element list pointed to by the phrase “at least one”, whether or not it is associated with a clearly identified element. It also allows you to do it.

  As a result, as a non-limiting example, “at least one of A and B” (or, in the same sense, “at least one of A or B”, or in the same sense “at least one of A and / or B” One "), in one embodiment, includes at least one, optionally one or more, A, and B is absent (and optionally includes elements other than B); in another embodiment, at least One, optionally including one or more B, A not present (and optionally including elements other than A); in yet another embodiment, at least one, optionally one or more A, and It may mean including at least one, optionally one or more B (and optionally including other elements), and the like.

  As used herein, all transitional phrases such as “comprising”, “including”, “carrying”, “having”, “containing”, “containing”, “ “Involving”, “holding” and the like are understood to be open-ended, ie, including but not limited to. Any composition and solution described herein shall be deemed to vary by any of these terms.

  Only the transitional phrases “consisting of” and “consisting essentially of” must be a closed or semi-closed transitional phrase, respectively, as indicated in the US Patent Office Examination Procedure. Don't be. “Consisting of” excludes any element, step, or material not specified. “Consisting essentially of” includes only the specified elements, steps, and materials and those that do not substantially affect the basic and new properties of the compositions and solutions described herein. . Any composition and solution described herein shall be considered to vary depending on either or both of these terms.

  A “biological sample” is, for example, a biological material collected from a cell, tissue, organ, or living body in order to detect an analyte. Typical biological samples include body fluids, cells, or tissue samples. Examples of the body fluid include serum, blood, plasma, saliva, urine, sweat, or peritoneal fluid. Cell or tissue samples include tissue biopsies, tissues, cell suspensions, or other specimens and samples such as clinical samples.

  “Crystal” is one form of the solid state of matter and includes atoms arranged in a three-dimensional periodic repeating pattern (eg, Barret, Structure of 25 Metals, 2nd ed., McGraw-Hill, New York (1952)). The crystalline form of the polypeptide is different from, for example, the second form-amorphous solid state. Crystals exhibit properties including shape, lattice structure, percent solvent, and optical properties such as refractive index. The crystals can be in a crosslinked or non-crosslinked form.

  By “functional fragment” of an oxalate degrading enzyme is meant a portion of an oxalate degrading enzyme polypeptide that retains the biological activity of one or more enzymes, eg, the ability to catalyze the decarboxylation or oxidation of oxalate. As used herein, functional fragments can include truncation at one or both ends unless otherwise indicated. For example, a functional fragment can have 1, 2, 4, 5, 6, 8, 10, 12, 15, or 20 or more residues excluded from the amino and / or carboxyl terminus of the oxalate degrading enzyme polypeptide. . In some embodiments, the cleavage is no more than 20 amino acids from one or both termini. A functional fragment can optionally bind to one or more heterologous sequences.

  The term “patient”, “individual” or “subject” means any mammal, including but not limited to humans, non-human primates, primates, baboons, chimpanzees, monkeys, rodents (eg , Mouse, rat), rabbit, cat, dog, horse, cow, sheep, goat, pig and the like.

  The term “isolated” means a molecule that is substantially away from its natural environment. For example, an isolated protein is substantially separate from other proteins from cellular material or derived cell or tissue sources. The term refers to an isolated protein that is sufficiently pure or at least 70% to 80% (w / w) pure, more preferably at least 80% to 90% (w / w) pure, for administration as a therapeutic composition. Still more preferably 90-95% pure; and most preferably at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8% or 100% (w / w ) Means a preparation that is pure.

  A unit dose is defined as the amount of enzyme that degrades 1 microgram of substrate per minute at 37 ° C. For example, a unit dose of oxalate decarboxylase is the amount of oxalate decarboxylase that degrades 1 microgram of oxalate per minute at 37 ° C.

  Conditions associated with increased concentrations of metabolites include, but are not limited to, oxalate-related disorders, uric acid-related disorders, urea-related disorders, bilirubin-related disorders, and phenylalanine-related disorders. Such conditions and disorders can optionally be acute or chronic.

  As used herein, “oxalate-related disorder” means a disease or disorder associated with a pathological concentration of oxalate or oxalate, including but not limited to oxalate, hyperoxalate, hyperoxalate. Aciduria, primary hyperoxaluria, intestinal hyperoxaluria, idiopathic hyperoxaluria, end-stage renal disease, ethylene glycol (oxalate) poisoning, idiopathic urolithiasis, renal failure (progressive, chronic, or (Including end stage renal failure), seborrhea, malabsorption, ileal disease, vulva pain, cardiac conduction disorder, inflammatory bowel disease, cystic pulmonary fibrosis, pancreatic exocrine insufficiency, Crohn's disease, ulcerative colitis, renal calcification Urinary stones, and nephrolithia. Such conditions and disorders can optionally be acute or chronic. Oxalate-related disorders associated with the kidney, bone, liver, gastrointestinal tract, and pancreas are known in the art. In addition, calcium oxalate can deposit in a wide variety of tissues and cause many oxalate-related disorders including, but not limited to, eyeballs, blood vessels, joints, bones, muscles, heart and other major organs It is well known that there is sex.

"Oxalic acid" exists mainly in its salt form, oxalate (as the salt of the corresponding conjugate base) at the pH of urine and intestinal fluid (pK _a1 = 1.23, pK _a2 = 4.19). Earnest, Adv. Internal Medicine 24: 407 427 (1979). The terms “oxalic acid” and “oxalate” are used interchangeably throughout this publication. Oxalate salts containing lithium, sodium, potassium, and iron (II) can be soluble, but calcium oxalate can be very poorly water soluble (eg, about 0.58 mg / 100 ml at 18 ° C. Earnest, Adv. Internal Medicine 24: 407 427 (1979), which only dissolves, oxalic acid from food may mean food oxalate. Oxalates produced by metabolic processes can mean endogenous oxalates. The circulating oxalate can be an oxalate present in a circulating fluid, such as blood.

  The term “therapeutically effective dose” or “therapeutically effective amount” reduces and prevents the concentration of metabolites in the blood, delays the onset of symptoms, or conditions such as, but not limited to, oxalic acid, high oxalic acid Means the amount of metabolic enzymes or functional fragments thereof that can include amelioration of symptoms of oxalate-related conditions, including hypertension and hyperoxaluria, such as primary hyperoxaluria or intestinal hyperoxaluria. In a therapeutically effective amount, for example, treating a disorder associated with increased oxalate concentration, preventing the disorder, reducing the severity of the disorder, delaying the onset of the disorder, and / or one of the disorders The risk of developing the above symptoms can be reduced.

The terms dialysate “dialysis solution”, “dialysate” and “dialysate” are used interchangeably herein. Dialysis solution means an aqueous solution having a suitable solute at a concentration suitable for peritoneal dialysis. Examples of peritoneal dialysis include continuous portable peritoneal dialysis (CAPD), intermittent peritoneal dialysis (IPD), continuous periodic peritoneal dialysis (CCPD), automatic peritoneal dialysis (APD), continuous flow peritoneal dialysis (CFPD). ), Regenerative peritoneal dialysis (RPD), and continuous flow regenerative peritoneal dialysis (CFRPD).

  In some embodiments, the dialysis solution used for peritoneal dialysis can include an aqueous solution comprising at least one osmotic agent, at least one electrolyte, and at least one organic acid salt. In some embodiments, the osmotic agent can be selected from, for example, glucose, icodextrin, and amino acids. In some embodiments, the electrolyte can be selected from, for example, sodium, potassium, calcium, and magnesium. In some embodiments, the organic acid salt can be selected from, for example, lactate, bicarbonate, and lactate. The pH of the peritoneal dialysis solution described herein can be in the range of 5-8. In some embodiments, the pH range can vary from 5.2 to 7.4, such as 6.0 to 7.4, and 7.0 to 7.4. In some embodiments, the peritoneal dialysis solution described herein can further include a bioactive agent that decreases oxalate concentration or increases the activity or effectiveness of metabolic enzymes. The components of the peritoneal dialysis solution described herein include control of electrolyte concentration or acid-base balance, removal of waste material, effective ultrafiltration, and / or metabolic enzymes described herein. Can be selected to manipulate the activity of.

  The peritoneal dialysis solution described herein can be selected from single-chamber and multi-chamber dialysis solutions. Multi-chamber dialysis solutions are known in the art, for example those described in WO 99/27885. As a result, various solutes can be retained in the separate compartment, among other things, to adjust the concentration of the active ingredient in the final conditioning solution. Peritoneal dialysis solutions are available from commercial suppliers such as Baxter (eg, PHYSIONEAL ™, DIANAL ™, EXTRANEAL ™, or NUTRINEAL ™), Gambro (eg, GAMBROSOLTRIO ™), and Fresenius ( For example, it can be purchased from BALANCE (trademark) or BICAVERA (trademark). Various types of peritoneal dialysis solutions and methods for their preparation are known in the art. For example, de Vin et al. Perit. Dial. Int. 29: 5-15 (2009); Schmitt et al. , Pediatr. Nephrol. 26: 1137-47 (2011); Garcia-Lopez et al. Nat. Rev. Nephrol. 8: 224-33 (2012).

  In some embodiments, the peritoneal dialysis solution can further include an agent to facilitate peritoneal dialysis, such as a surfactant or wetting agent (see, eg, Penzotti et al., J Pharm Sci. 57 ( 7): 1192-95 (1968)). In some embodiments, the wetting agent can be anionic, cationic, or nonionic. In certain embodiments, the wetting agent is sodium dioctyl sulfosuccinate. In some other embodiments, the agent for promoting peritoneal dialysis is one selected from catecholamines such as norepinephrine and dopamine (eg, Hirzel et al., J Lab Clin Med. 94 (5 ): 747-54 (1979)). In certain embodiments, the agent is dopamine.

  As used herein, metabolic enzyme means an enzyme capable of reacting with a product from human or animal metabolism, for example, an enzyme capable of decomposing uric acid, urea, or oxalate.

Urate oxidase (uricase)
The terms “uric acid oxidase” and “uricase” are used interchangeably herein. Urate oxidase (uricase; EC 1.7.3.3) means urate oxidase enzyme. Urate oxidase includes a well-defined group of enzymes that can catalyze uric acid to oxidize into more soluble products, allantoin, and more easily excreted purine metabolites. .

Uric acid + O ₂ + H ₂ O → 5-hydroxyisouric acid + H ₂ O ₂ → Allantoin + CO ₂

  Increased uric acid levels in the blood can cause hyperuricemia and gout. Included in this definition are isoforms of urate oxidase and glycoforms of these isoforms. Uric acid oxidases from plants, bacteria and fungi are encompassed by the term and include true urate oxidases from bacteria and fungi, such as yellow Aspergillus. In certain circumstances, urate oxidase can be a soluble or insoluble tetrameric protein. The cDNA encoding this protein can be obtained from Escherichia coli (Legoux et al., J. Biol. Chem., 267: 8565-8570 (1992)), yellow Aspergillus (Chevalet et al., Curr. Genet. 21: 447-453). 1992)), and Saccharomyces cerevisiae (Leplatois et al., Gene 122, 139-145 (1992)). Examples of the genetically modified urate oxidase include urate oxidase produced by a genetically modified microorganism, and can be obtained from, for example, the above-described genetically modified strains of Escherichia coli and Saccharomyces cerevisiae. Rasburicase refers to a recombinant urate oxidase enzyme produced from a Saccharomyces cerevisiae yellow transgenic strain cloned with cDNA from an Aspergillus strain (Oldfield et al., Drugs 66 (4): 529-545 (2006), Leplatois et al., Gene 122: 139-145 (1992)). Rasburicase includes a tetrameric protein having approximately the same molecular mass of each subunit of about 34 kDa as that of natural yellow Aspergillus urate oxidase (Bayol et al., Biotechnol. Appl. Biochem. 36: 21-31). 2002).

Urease urease (EC 3.5.1.5) refers to the urease enzyme. Ureases include a well-defined group of enzymes that can catalyze the hydrolysis of urea into carbon dioxide and ammonia.

(NH ₂ ) ₂ CO + H ₂ O → CO ₂ + 2NH ₃
High concentrations of urea cause uremia and are often accompanied by kidney failure. Urease isoforms and glycoforms of these isoforms are included in this definition. Plants, urease from bacteria and fungi are encompassed by the term, intrinsic urease from bacteria and fungi, for example, Ureaplasma urealyticum, Actinomyces naeslundii, Yersinia pestis, Filobasidiella neoformans, Coccidioides immitis, Bordetella bronchiseptica, Streptococcus salivarius, Mycobacterium tuberculosis , Actinobacillus pleuropneumoniae, thermophilic Bacillus, Ureaplasma urealyticum, Ureaplasma urealyticum, Yersinia ps udotuberculosis, Canavalia ensiformis, Bacillus pasteurii, Heliobacter heilmannii, Yersinia enterocolitica, Pmirabilis, Kiebsiella aerogenes, Kiebsiella pneumonia, Helicobacter pylori, and Escherichia coli can be mentioned.

  As used herein, oxalate degrading enzyme means an enzyme that can catalyze the degradation of oxalate. In some embodiments, the oxalate degrading enzyme can include, for example, oxalate oxidase, oxalate decarboxylase, oxalyl-CoA decarboxylase, and formyl CoA transferase. For example, Svedruzic et al. , Arch Biochem. Biophys. 433: 176-92 (2005).

Oxalate oxidase As used herein, oxalate oxidase (OXO) (EC 1.2.3.4) refers to the oxalate: oxygen oxidoreductase enzyme. Oxalate oxidase includes a well-defined group of enzymes that can catalyze oxalate to molecular oxygen (O ₂ ) dependent oxidation to carbon dioxide and hydrogen peroxide according to the following reaction: .

HO ₂ C—CO ₂ H + O ₂ → 2CO ₂ + H ₂ O ₂
Oxalate oxidase isoforms and glycoforms of these isoforms are included in this definition. OXOs from plants, bacteria and fungi are encompassed by the term and include true cereal OXOs such as wheat, barley, corn, oats, rice, and rye. These enzymes can be identified as germin-type OXO (G-OXO) because wheat oxalate oxidase is also known as germin. Germin-like proteins (GLPs) include a broad class of proteins that share certain structural features. Other OXO sources include cedar, beet, spinach, sorghum, and banana. OXO, such as G-OXO, can be as active as, for example, a hexameric glycoprotein. Optionally, OXO can be further superoxide dismutase activity, such as barley OXO. In certain circumstances, OXO can be a soluble hexameric protein, including trimers of OXO glycoprotein dimers.

Oxalate decarboxylase As used herein, oxalate decarboxylase (OXDC) (EC 4.1.1.2) refers to the oxalate carboxylase enzyme. Oxalate decarboxylase is a group of enzymes known in the art that can catalyze oxalates to molecular oxygen (O ₂ ) independent oxidation to carbon dioxide and formate according to the following reaction: Is mentioned.

_{HO 2 C-CO 2 H →} 1CO 2 + HCOOH
Oxalate decarboxylase isoforms and glycoforms of these isoforms are included in this definition. OXDC from plants, bacteria and fungi are encompassed by the term, and true oxalate decarboxylase from fungi and fungi, such as Bacillus subtilis, Collibia velutipes or Flammulina velutipes, Aspergillus niger, Pseudomonas. Synechocystis sp. Streptococcus mutans, Trametes hirsute, Sclerotinia sclerotiorum, T. et al. Versicoror, Postia placenta, Myrothium verrucaria, Agaricus bisporus, Methylobacterium extratropis, Pseudomonas oxalaticus, Ralstonia europhus. Oxalibacterium flavum, Ammoniiphilus oxalaticus, Vibrio oxalaticus, A. oxalativorans, Variovorax paradoxus, Xanthobacter autotropicus, Aspergillus sp. , Penicillium sp. , And Mucor specials, OXDC is further dependent on coenzyme A, for example, OXDC from intestinal organisms. In certain circumstances, OXDC can be a soluble or insoluble hexameric protein.

Oxalyl-CoA decarboxylase and formyl CoA transferase As used herein, oxalyl-CoA decarboxylase (OXC) (EC 4.1.1.8) refers to the oxalyl-CoA decarboxylase enzyme. Formyl-CoA transferase (FRT) (EC 2.8. 3.16) refers to the formyl-CoA transferase enzyme. Oxalyl-CoA decarboxylase includes a group of enzymes known in the art that can catalyze oxalyl-CoA to formyl-CoA and carbon dioxide.

Formyl-CoA transferase includes a group of enzymes known in the art that can exchange formate and oxalate with CoA.

As a result, oxalate can be converted to formate and carbon dioxide by the binding action of oxalyl-CoA decarboxylase and formyl-CoA transferase. OXC and FRC isoforms, and glycoforms of these isoforms are included in this definition. OXC and FRC from plants, bacteria and fungi are encompassed by the term and include enzymes from bacteria and fungi such as Pseudomonas oxalaticus and Oxalacterformigenes. In certain circumstances, OXC can be a soluble or insoluble tetrameric protein.

Bilirubin oxidase bilirubin oxidase (BO) (EC 1.3.3.5) refers to an enzyme that catalyzes the reaction of oxidizing bilirubin to biliverdin and multi-copper oxidase (having multiple copper ions in the active center) It is a kind of enzyme that falls under the category of “general term of enzyme”. The enzyme catalyzes the chemical reaction.

In addition, bilirubin oxidase M-1, which is involved in the metabolism of porphyrin and chlorophyll, is mentioned as the enzyme. Tanaka et al. Agric. Biol. Chem. 49: 843-844 (1985). Hyperbilirubinemia occurs when the concentration of bilirubin in the blood is higher than normal and is also related to jaundice. Included in this definition are BO isoforms and the glycoforms of these isoforms. BO from plant bacteria and fungi is encompassed by the term and is, for example, an enzyme from Myrothecerium verrucaria. Mizutani et al. Acta Cryst. F66 (7): 765-770 (2010); Cracknell et al. Dalton Trans. 40 (25): 765-770 (2011).

Phenylalanine hydroxylase Phenylalanine hydroxylase (PAH, PheOH, or PheH) (EC 1.14.16.1) hydroxylates the aromatic side chain of phenylalanine, a rate-limiting step in Phe catabolism, to produce tyrosine It means an enzyme that acts as a catalyst. The brain is sensitive to Phe concentrations, and lack of PAH enzyme can result in excessive Phe concentrations or hyperphenylalaninemia. Insufficient PAH enzyme activity is in the range of moderately elevated plasma Phe with unknown clinical outcome, due to the potential of classic phenol ketoneuria (PKU) and severe central nervous system dysfunction (intellectual developmental disorder) There is a possibility. Lack of PAH enzyme activity is the most common cause of hyperphenylalaninemia. The human PAH gene span 90 kb consists of 13 exons and is localized to chromosome 12q24.1. Lidsky et al. Proc Natl Acad Sci USA, 82: 6221-6225 (1985); DiLella et al. , Biochemistry, 25: 743-749 (1986). The structure and function of human PAH has been studied. Hufton et al. Biochem J, 311: 353-366 (1995); Waters et al. , Hum Mutat. 11: 4-17 (1998). PAH isoforms and glycoforms of these isoforms are included in this definition. PAH from other sources is included in the term.

Phenylalanine ammonia lyase Phenylalanine ammonia lyase (PAL; EC 4.3.1.5.) Refers to an enzyme that catalyzes the reaction of converting L-phenylalanine to ammonia and trans-cinnamic acid. Such enzymes may play a role in the treatment and diagnosis of phenorketonuria (PKU). Ambrus et al. , Science, 201: 837-839 (1978). PAL is a microorganism, especially fungi, such as Rhodotorula sp. Rhodos-oridium sp. Sporobolus sp. Geotrichum sp. , Moniliella sp. Pellicularia sp, Gonatobotryum sp. , Synchalalastrum sp. , Endomyces sp. , Aspergillus sp. Saccharomvcopsis sp. , Eurotium sp. Glomerella sp. , Cladosporium sp. It may also be derived from Trichosporon sp and may be derived from plants such as Pisum sativum, potato, sweet potato or soybean.

Isolated Metabolic Enzymes The metabolic enzymes used to prepare the peritoneal dialysis solution of the present invention can be isolated, for example, from natural sources or derived from natural sources. As used herein, the term “derived from” means having an amino acid or nucleic acid sequence that naturally appears in the source. For example, barley-derived oxalate oxidase can include the primary sequence of barley oxalate oxidase protein, or can be encoded by a nucleic acid that includes a sequence found in barley that encodes oxalate oxidase or a modification thereof. . The oxalate decarboxylase derived from Bacillus subtilis can include the primary sequence of a Bacillus subtilis oxalate decarboxylase protein, or is encoded by a nucleic acid that includes a sequence found in Bacillus subtilis that encodes oxalate decarboxylase or a variant thereof. can do. The oxalyl-CoA decarboxylase or formyl CoA transferase derived from Oxalobacter formigenes can comprise the primary sequence of the Oxalacter formigenes oxalyl-CoA decarboxylase or formyl CoA transferase protein, or oxalyl-CoA decarboxylase or formyl CoA transferase, or It can be encoded by a nucleic acid comprising a sequence found in Oxalacter formigenes encoding the alteration. Proteins or nucleic acids derived from a source include molecules isolated from the source, molecules produced by genetic recombination, and / or chemically synthesized or denatured molecules. The compositions provided herein can include a polypeptide comprising an amino acid sequence of a metabolic enzyme or an amino acid sequence from a functional fragment of the enzyme that retains oxalate degradation activity. In some embodiments, the enzyme is a functional property of at least one natural enzyme, such as the ability to catalyze the degradation of oxalate, the ability to multimerize, an ion (eg, manganese) requirement, and / or Other catalytic capabilities (eg, superoxide dismutase activity) can be retained.

  Oxalate oxidase has been previously isolated and as a result is available from a number of sources including barley seedlings, roots and leaves, beet stalks, beet leaves, tapas, sorghum leaves, and banana peel. OXO can also be purchased from commercial suppliers, such as Sigma. Methods for isolating OXO from natural sources have been previously described, for example, in the following references: Liu et al. , Zhi Wu Sheng Li Yu Fen Zi Sheng Wu Xue Xue Bao 30: 393-8 (2004) (Engl. Abst. At PMID 15627687); Rodriguiez-Lopez et al. , FEBS Lett. 9: 44-48 (2001); Pundir et al. , Chin. J. et al. Biotechnol. 15: 129-138 (1999); and Agillar et al. , Arch. Biochem. Biophys. 366: 275-82 (1999). These isolated oxalate oxidases can be used to form the compositions described herein.

  Oxalate decarboxylase is pre-isolated, as a result, Bacillus subtilis, Collybia velutipes or Flammulina velutipes, Aspergillus niger, Pseudomonas sp, Synechocystis sp, Streptococcus mutans, Trametes hirsute, Sclerotinia sclerotiorum, T versicolor, Postia placenta, Myrothecium verrucaria , Agaricus bisporus, Methylobacterium extorquens, Pseudomonas oxalaticus, Ralstonia eutr pha, Cupriavidus oxalaticus, Wautersia sp, Oxalicibacterium flavum, Ammoniiphilus oxalaticus, Vibrio oxalaticus, A oxalativorans, Variovorax paradoxus, Xanthobacter autotrophicus, Aspergillus sp. , Penicillium sp. , And from a number of sources including Mucor specifications. OXDC can also be purchased from commercial suppliers such as Sigma. Methods for isolating OXDC from natural sources have been previously described, for example, in the following references: Tanner et al. , The Journal of Biological Chemistry. 47: 43627-43634. (2001); Dasek et al. , Methods in plant biochemistry and molecular biology. Boca Raton, FL: CRC Press. 5: 49-71. (1997); Magro et al. , FEMS Microbiology Letters. 49: 49-52. (1988); Anand et al. Biochemistry. 41: 7659-7669. (2002); and Tanner and Bornemann, Journal of Bacteriology. 182: 5271-5273 (2000). These isolated oxalate decarboxylases can be used to form the compositions described herein.

  Oxalyl-CoA decarboxylase and formyl CoA transferase were previously isolated, so that oxalate-degrading bacteria, Pseudomonas oxalaticus present in soil (Qyayl et al., Biochem. J. 78: 611-615 (1961)) and Available from a number of sources, including Oxalacterformigenes, humans (Allison et al., Arch. Microbiol. 141: 47 (1985)) present in the vertebrate gastrointestinal tract. OXC and FRT can also be purchased from commercial suppliers such as Sigma. In addition, enzymes for oxalate formate exchange and membrane transporters have been both purified and characterized (Baetz and Allison, J. Bact. 171: 2605-2608 (1989); Baetz and Allison). , J. Bact. 172: 3537-3540 (1990); Ruan et al., J. Biol. Chem. 267: 10537-19543 (1992)). These isolated oxalyl-CoA decarboxylase and / or formyl CoA transferase can be used to form the compositions described herein.

Genetically modified metabolic enzymes or genetically modified metabolic enzymes can be used to form the compositions provided herein. In some embodiments, the recombinant metabolic enzyme can include a sequence from a natural enzyme sequence or be encoded by a sequence from a natural enzyme sequence. In addition, the metabolic enzymes include amino acid sequences that are homologous to, or substantially identical to, the natural sequences described herein. Also provided are metabolic enzymes encoded by nucleic acids that are homologous or substantially identical to nucleic acids encoding natural enzymes and can be used as described herein.

  Polypeptides referred to herein as “genetically modified” include polypeptides produced by genetically modified DNA methods, such as artificial recombination methods such as synthetic enzyme chain reaction (PCR) and / or restriction enzymes. Polypeptides produced by methods that rely on cloning into vectors using are included. “Genetical recombination” polypeptides also include polypeptides that have altered expression, eg, natural polypeptides that have been modified by genetic recombination in a cell, eg, a host cell.

  In some embodiments, OXO can be genetically engineered from nucleic acid homologous to the barley OXO nucleic acid sequence, sometimes to increase or optimize genetically engineered production, eg, in a heterologous host. Can be modified. Examples of such modified sequences include the barley OXO open reading frame nucleic acid sequence. The OXO sequence can be modified to reduce its GC content, is bound to the conjugating factor secretion signal sequence, and is tangent at the recombinant restriction endonuclease cleavage site. In some embodiments, OXO can be generated recombinantly from an unmodified barley nucleic acid sequence available at GenBank Accession No: L15737.

  In some embodiments, OXDC can be genetically engineered from nucleic acid that is homologous to a Bacillus subtilis or Collibia velutepes OXDC nucleic acid sequence, and sometimes increases, for example, genetically engineered production in a heterologous host. Or it can be modified to optimize. An example of such a modified sequence is the nucleic acid sequence of the open reading frame of the OXDC of Colibia velocipes for expression in Candida bodyini. The OXDC sequence can be modified to reduce its GC content, is bound to the conjugation factor secretion signal sequence, and is tangent at the recombinant restriction endonuclease cleavage site. In some embodiments, OXDC can be genetically engineered from an unmodified Bacillus subtilis OXDC nucleic acid sequence available at GenBank Accession No: Z99120.

  In some embodiments, the OXC or FRT can be generated by genetic recombination from nucleic acids that are homologous to the OXC or FRT nucleic acid sequence of Pseudomonas oxalaticus or Oxalacterformigenes, and sometimes, for example, genetically modified in a heterologous host. Modifications can be made to increase or optimize production. In some embodiments, OXCs can be generated recombinantly from OXC nucleic acid sequences of Oxalacter formigenes. In some embodiments, the FRT can be generated by genetic recombination from an Oxalacterformigens FRT nucleic acid sequence.

  In addition, the genes of all three proteins are cloned, sequenced and expressed as biologically active recombinant proteins (Abe et al., J. Biol. Chem. 271: 6789-6793 (1996); Lung et al., J. Bact. 179: 3378-3381 (1994); Sidhu et al., J. Bact. 179: 3378-3381 (1997)).

  In some embodiments, a metabolic enzyme useful in forming the compositions described herein comprises a nucleic acid construct comprising a coding sequence for a host cell, e.g., a metabolic enzyme polypeptide or functional fragment thereof. It can be expressed in the host cell it contains. Suitable host cells for expression of the enzyme include, for example, yeast, bacteria, fungi, insects, plants, or mammalian cells, or transgenic plants, transgenic animals, or cell-free systems. it can. In some embodiments, the host cell can glycosylate the enzyme polypeptide, can disulfide bond, can secrete the enzyme, and / or multiple enzyme polypeptides if necessary. It can be one that can help quantification. Exemplary host cells include, but are not limited to, E. coli. cells (including E. coli Origami B and E. coli BL21), Hansenula polymorpha, Pichia pastoris, Saccharomyces cerevisiae, Schucharacies pombe, Bacillus sp. Kidney), and other human cells. In addition, transgenic plants and animals, including pigs, cows, sheep, horses, chickens, and rabbits, can be suitable hosts for the production of the enzymes described herein.

  Genetic production of a metabolic enzyme may include a configuration in the form of a plasmid, vector, phagemid, or transcription or expression cassette in which the host or host cell contains at least one nucleic acid encoding the metabolic enzyme or functional fragment thereof. it can. A variety of configurations are available, including configurations that are maintained in a single copy or multiple copies, or that are integrated into the host cell chromosome. Many recombinant expression systems, components, and reagents for recombinant expression are described, for example, in Invitrogen Corporation (Carlsbad, Calif.); S. Biological (Swampscott, Mass.); BD Biosciences Pharmingen (San Diego, CA); Novagen (Madison, WI); Stratagene (La Jolla, Gur gen Gur, M It is commercially available.

  In some embodiments, genetic expression of metabolic enzymes can be controlled by heterologous promoters, optionally including constitutive and / or inducible promoters. For example, T7, alcohol oxidase (AOX) promoter, dihydroxyacetone synthase (DAS) promoter, Gal1, 10 promoter, phosphoglycerate kinase promoter, glyceraldehyde-3-phosphate dehydrogenase promoter, alcohol dehydrogenase promoter, copper metallothionein (CUP1) promoter Promoters such as acid phosphatase promoter, CMV and promoter polyhedrin may also be preferred. The particular promoter can be selected based on the host or host cell. Also, for example, promoters that can be induced by methanol, copper sulfate, galactose, low phosphoric acid, alcohols such as ethanol can be used and are well known in the art.

  In some embodiments, the nucleic acid encoding the metabolic enzyme can optionally include a heterologous sequence. In some embodiments, for example, the secretory sequence can be included at the N-terminus of the enzyme polypeptide. Secretion derived from conjugating factor, BGL2, yeast acid phosphatase (PHO), xylanase, signal sequence from alpha amylase, signal sequence from other yeast secreted proteins, and other species capable of inducing secretion from the host cell A signal sequence such as a signal peptide may be useful. Similarly, other heterologous sequences such as linkers (eg, containing cleavage or restriction endonuclease sites) and one or more expression control elements, enhancers, terminators, leader sequences, and one or more translation signals are described herein. Within range. These sequences can optionally be included in the configuration and / or linked to the nucleic acid encoding the enzyme. Unless otherwise indicated, “linked” sequences can be directly or indirectly linked to each other.

  Similarly, an epitope or affinity tag, such as histidine, HA (hemagglutinin peptide), maltose binding protein, AviTag®, FLAG, or glutathione-S-transferase can optionally be linked to the enzyme polypeptide. The tag can optionally be cleavable from the enzyme after production or purification. One skilled in the art can readily select a suitable heterologous sequence, eg, a suitable host cell, composition, promoter, and / or secretory signal sequence.

  Enzyme homologues or variants may differ from the enzyme reference sequence by one or more residues. For example, structurally similar amino acids can be used in place of some specific amino acids. Structurally about the same amino acids include, for example, (I, L and V); (F and Y); (K and R); (Q and N); (D and E); and (G and A) Can be mentioned. Amino acid deletions, additions, or substitutions are also encompassed by the enzyme homologs described herein. Such homologues and variants include, for example, (i) polymorphic variants and natural or artificial variants, (ii) modified polypeptides in which one or more residues are modified, and (iii) Mutants that contain one or more modified residues are included.

  The enzyme polypeptide or nucleic acid is at least 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the reference sequence Are “homologous” (or “homologous”). If the homolog is not identical to the reference sequence, it is a “variant”. The homologous nucleotide or amino acid sequence differs from the reference sequence (eg, by truncation, deletion, substitution, or addition) by only 1, 2, 3, 4, 5, 8, 10, 20, or 50 residues And a homologue is “substantially identical” to a reference enzyme sequence if it retains the ability to catalyze the degradation of enzymatic substrates (or encodes a polypeptide that retains this ability). A fragment of a metabolic enzyme comprising a variant and / or a substantially identical sequence can be a homolog. By way of example, a homologue can be derived from various sources of an enzyme, or a homologue can be derived from or related to a reference sequence by truncation, deletion, substitution, or addition mutation. Can do. The percent identity between two nucleotide or amino acid sequences is determined by standard alignment algorithms, such as Altschul et al. , J Mol. Biol. 215: 403 410 (1990), the algorithm of Needleman et al. , J .; Mol. Biol. 48: 444 453 (1970), or the algorithm of Meyers et al. , Comput. Appl. Biosci. 4:11 17 (1988), and can be revealed by the Basic Local Alignment Tool (BLAST). Such an algorithm is incorporated into the BLASTN, BLASTP and “BLAST 2 Sequence” programs (reviewed in McGinnis and Madden, Nucleic Acids Res. 32: W20-W25, 2004). When using such a program, default parameters can be used. For example, for nucleotide sequences, the following settings can be used for “BLAST2 sequence”: program BLASTN, reward for match 2, penalty for missmatch 2, open gap and extension gap penalties are 5 and 2, respectively, gap x_dropoff 50, expect 10, word size 11, and filter ON. For amino acid sequences, the following settings can be used for the “BLAST2 sequence”: programs BLASTP, matrix BLOSUM62, open gap and extension gap penalties 11 and 1, respectively, gap x_dropoff50, expect 10, word size 3. Suitable amino acid and nucleic acid sequences for metabolic enzymes suitable to form the compositions described herein can include homologous, variant, or substantially identical sequences. In some embodiments, the enzyme homologue is at least 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% relative to the reference sequence. , 99%, or 100% activity.

Purification of Metabolic Enzymes A protein or polypeptide of an enzyme described herein can be purified from a source, eg, a natural or genetically modified source, and a polypeptide referred to herein as “isolated” Substantially its natural environment, eg
A polypeptide that is remote from the protein, lipid, and / or nucleic acid (eg, cell, tissue (eg, plant tissue), or fluid or medium (in the case of a secreted polypeptide)) of its origin. Isolated polypeptides include those obtained by the methods described herein or other suitable methods, polypeptides that are substantially pure or essentially pure, and chemical synthesis, genetically produced products Or a polypeptide purified by a combination of biological and chemical methods. Optionally, the isolated protein can be further processed after its production step, eg, purification step.

  In some embodiments, purification can include buffer exchange and chromatography steps. Optionally, a concentration step, such as dialysis, a step by chromatofocusing chromatography and / or a step with buffer exchange can be used. In certain embodiments, Q-Sepharose, DEAE Sepharose, DE52, sulfopropyl sepharose chromatography or cation or anion exchange chromatography including CM52 or similar cation exchange column can be used for purification. Buffer exchange is optionally performed prior to chromatographic separation and can be performed by tangential flow filtration, eg, diafiltration.

  In certain preparations, the metabolic enzyme is at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99. It can be 9% pure.

  In some embodiments, purification performed on a gram scale may be preferred for the preparation of metabolic enzymes, and the method can be optimized for efficient and inexpensive production scale enzyme purification. The purification method provides, for example, purification of at least 0.5, 1, 2, 5, 10, 20, 50, 100, 500, or 1000 grams or more of metabolic enzyme. One exemplary method provides tangential flow filtration of at least 10 L, 50 L, 100 L, 500 L, 1000 L or more of the starting sample, allowing buffer exchange and precipitation of contaminating proteins. A single Q-Sepharose column can optionally be used to purify the enzymes described herein.

  In some embodiments, a metabolic enzyme described herein, eg, an oxalate degrading enzyme, can be soluble or crystalline. Enzyme crystals can be prepared and / or dried using methods known in the art (see, eg, WO 2006/135926 and WO 2008/105911). In some embodiments, the enzyme may not crystallize. In some embodiments, the enzyme can be covalently modified, eg, PEGylated. Examples of the pegylation method include US Patent No. See 6,783,965. In some embodiments, the enzyme can be cross-linked. In some other embodiments, the enzyme may not be cross-linked. In some embodiments, the enzyme can be a crosslinked crystal (see, eg, US Patent No. 6,004,768).

Methods of Treating Metabolite-Related Disorders The methods of the present invention include a mammalian subject to treat, prevent, or reduce the risk of developing conditions associated with high concentrations of metabolites, such as oxalate. In some embodiments, which can comprise administering a peritoneal dialysis solution described herein in a therapeutically effective amount, the method prepares an additional peritoneal dialysis solution comprising at least one metabolic enzyme; and Administration of an additional peritoneal dialysis solution to the mammalian subject can be included. Increased concentrations of metabolites, such as oxalate, can be detected in a biological sample from a subject, such as a body fluid, including, for example, urine, blood, serum, plasma, or peritoneal fluid. In certain embodiments, urine or serum oxalate concentration can be detected. In some embodiments, the metabolite is selected from oxalate, uric acid, urea, bilirubin, and phenylalanine. In some embodiments, the metabolite is oxalate.

  In some embodiments, for example, oxalic acid, hyperoxalicemia, primary hyperoxaluria, enteral hyperoxaluria (including hyperoxaluria, eg, brought about by surgical treatment), idiopathic A method of treating an individual with high oxalic aciduria is provided. In other examples, the methods disclosed herein provide secondary to oxalate and / or disorders associated with elevated oxalate in the kidney (including, for example, end stage renal disorder), bone, liver gastrointestinal tract, and pancreas. May be easy to treat typical hyperoxalicemia. Further, disorders or diseases to be treated by the methods provided herein include, but are not limited to, oxalic acid and / or the following conditions: ethylene glycol (oxalate) addiction, idiopathic urolithiasis, kidney Failure (including progressive, chronic, or end-stage renal failure), seborrhea, malabsorption, ileal disease, vulva, inflammatory bowel disease, cystic pulmonary fibrosis, exocrine pancreatic failure, Crohn's disease, ulcerative colitis, Mention may be made of nephrocalcinosis, osteoporosis, urolithiasis and hyperoxalicemia secondary to nephrolithiasis. Such conditions and disorders can optionally be acute or chronic. In some embodiments, the disorder is associated with increased levels of uric acid, urea, bilirubin, or phenylalanine. In some embodiments, the disorder is selected from hyperuricemia, gout, uremia, hyperbilirubinemia, jaundice, hyperphenylalaninemia, and phenorketonuria (PKU). In certain embodiments, the methods described herein can be combined with other methods of reducing mammalian metabolite concentrations, such as hemodialysis.

  In some embodiments, the methods of the present invention allow the concentration of a subject's metabolite, eg, oxalate, to be at least 10%, 15%, 20%, 25% relative to the concentration of an untreated or control subject. , 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more. In some embodiments, the decrease is measured by comparing the concentration of the metabolite in the subject before and after administration of the composition. In some embodiments, the publication treats or ameliorates a metabolite-related condition or disorder to at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more, methods of ameliorating one or more symptoms of a condition or disorder are provided. In certain embodiments, the method can reduce endogenous oxalate concentration and / or food oxalate absorption.

  In some embodiments, individuals with genotypes associated with high oxalate concentrations, such as alanine, glyoxalate aminotransferase, glyoxalate reductase / hydroxypyruvate reductase, hepatic glycolate oxidase, dihydrodipicolinate synthase, or oxalate Methods are provided to treat homozygous or heterozygous individuals with mutations that reduce the activity of another enzyme involved in substance metabolism or another enzyme associated with hyperoxaluria. In other embodiments, a method of treating an individual with reduced or deficient intestinal colonization of Oxalacter formigenes is provided. See, for example, Hoppe et al. , Kidney Int. 70: 1305-11 (2006); Hoppe et al. , Am. J. et al. Kidney Dis. 58: 453-55 (2011).

  The disclosed methods can include treating a mammalian subject susceptible to or at risk of suffering from a condition associated with increased oxalate concentrations. Populations treated by the disclosed methods include, but are not limited to, oxalate-related disorders such as oxalate, hyperoxalate, primary hyperoxaluria, or intestinal hyperoxaluria. Include subjects who are or are at risk of developing

  Subjects to be treated according to the methods of the present invention include, but are not limited to, humans, non-human primates, primates, baboons, chimpanzees, monkeys, rodents (eg, mice, rats), rabbits, cats, Mammals include dogs, horses, cows, sheep, goats, pigs and the like.

  Many methods can be used to assess conditions associated with onset or advanced oxalate-related disorders or elevated oxalate concentrations. Such disorders include, but are not limited to, the conditions, diseases, or disorders as defined above. The onset or progression of an oxalate-related disorder can be assessed, for example, by measuring urine oxalate, plasma oxalate, measuring kidney or liver function, or detecting calcium oxalate deposits.

  For example, a condition, disease, or disorder can be identified by detecting or measuring the oxalate concentration of a urine sample or other biological sample or liquid. By detecting or measuring the oxalate concentration of a blood sample, for example, hyperoxalicemia can be identified. Early symptoms of hyperoxaluria can include, for example, kidney stones due to oxalic acid, with severe or sudden abdominal or flank pain, hematuria, frequent urination, pain during urination, or fever and chills there is a possibility. Kidney stones can be symptomatic or non-symptomatic and can be visualized, for example, by imaging the abdomen by X-ray, ultrasound, or computer tomography (CT) scan. If hyperoxaluria is not suppressed, the kidney is damaged and kidney function is impaired. There is even the possibility of kidney failure.

  Kidney failure due to decreased or insufficient urine output (reduced glomerular filtration rate), general fatigue, and significant malaise, nausea, vomiting, anemia, and / or failure of normal development and growth in children (And poor kidney function) can be identified.

  Calcium oxalate deposition of other tissues and organs by methods including direct visualization (eg, of the eyeball), X-ray, ultrasound, CT, echocardiography, or tissue biopsy (eg, bone, liver, or kidney) Objects can also be detected. Renal and liver function, and oxalate concentrations can also be assessed using direct and indirect analytical methods recognized in the art. The chemical content of urine, blood or other biological samples can also be tested by well known techniques. For example, oxalate, glycolate, and glycerate concentrations can be measured. Methods for analysis of liver and kidney function are well known, such as analysis of liver tissue for enzyme deficiency and analysis of kidney tissue oxalate deposits.

  Samples can also be tested for DNA changes known to cause various types of primary high oxalate.

  Other therapeutic applications can include the presence of one or more risk factors including, but not limited to, those described above and in the following sections. A subject at risk of developing or prone to developing a condition, disease, or disorder by confirming the presence or absence of one or more such risk factors, diagnostic, or prognostic indicators, or Subjects that may be receptive to treatment with the compositions described herein can be identified. Similarly, an individual at risk for developing an oxalate-related disorder can be identified by analyzing one or more genetic or expression markers.

  In some embodiments, the disclosed method has a urine oxalate concentration per 24 hours of at least 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 mg or more may be useful for subjects. In certain embodiments, the oxalate concentration can be associated with one or more symptoms or medical conditions. For example, the oxalate concentration can be measured in biological samples such as body fluids including blood, serum, plasma, or urine. Optionally, the oxalate can be converted to a standard protein or substance, eg, creatinine in urine. In some embodiments, the method comprises an undetected concentration or 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35% of the oxalate concentration prior to treatment of the subject. %, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or a composition described herein for reducing a subject's circulating oxalate concentration to less than 80% Administration may be included.

  Hyperoxalate and / or oxalate is often associated with various types of hyperoxaluria. Human high oxalate can be characterized by excretion of urinary oxalate in excess of 40 mg (about 440 μmol). A typical clinical cutoff concentration is, for example, 43 mg / day (about 475 μmol) for men and 32 mg / day (about 350 μmol) for women. Hyperoxaluria can also be defined as the excretion of urine oxalate with urine creatinine exceeding 30 mg per gram per day. Persons with mild high oxalate may excrete about 30-60 (about 342-684 μmol) or about 40-60 (about 456-684 μmol) mg of oxalate per day. For example, a person with intestinal hyperoxaluria may excrete about 45-80 mg (about 513-912 μmol) of urine oxalate per day, and a person with primary high oxalate may be at least 80 mg (about 912 μmol) per day. Urine oxalate may be excreted. Borowski et al. , Exp. Opinion Pharmacother. 7: 1887-96 (2006); Hoppe, Nat. Rev. Nephrol. 8: 467-75 (2012).

  Methods are provided for treating disorders associated with increased concentrations of mammalian metabolites, such as oxalate. The method can include administering to the mammal a peritoneal solution comprising a metabolic enzyme, eg, a metabolic enzyme described herein or a functional fragment thereof, in a therapeutically effective amount. In some embodiments, the method comprises preparing a peritoneal dialysis solution having at least one metabolic enzyme or functional fragment thereof, and administering the enzyme-added peritoneal dialysis solution to the mammal in a therapeutically effective amount. be able to. In some embodiments, the peritoneal dialysis solution can be administered by a peritoneal dialysis device, eg, a catheter. In certain cases, a peritoneal dialysis solution is administered to reduce endogenous or exogenous metabolite levels and / or concentrations. In some embodiments, the peritoneal dialysis solution reduces the metabolite level and / or concentration of the mammalian peritoneal cavity. In some embodiments, the metabolite is oxalate. In other embodiments, the metabolite is selected from uric acid, urea, bilirubin, and phenylalanine. In some embodiments, the disorder is selected from hyperuricemia, gout, uremia, hyperbilirubinemia, jaundice, hyperphenylalaninemia, and phenorketonuria (PKU). In certain embodiments, the methods described herein can be combined with other treatment methods for increasing metabolite concentrations, such as hemodialysis.

  Metabolic enzymes can be used to supplement the peritoneal dialysis solution as the sole active compound or in combination with another active compound or composition. The enzyme dose and duration of peritoneal dialysis can be selected based on the severity of the symptoms and the progression of the disease. An appropriate therapeutically effective dose of a metabolic enzyme can be selected by the treating clinician. Furthermore, specific doses shown in the Examples or known in the art can be used (eg, Physicians' Desk Reference (PDR) 2003, 57th ed., Medical Economics Company, 2002).

  In some embodiments, the dose of metabolic enzyme can be about 50, about 100, about 150, or about 200 unit dose per 100 ml of peritoneal dialysis solution. In some embodiments, the dose can be about 150 unit doses of enzyme per 100 ml of peritoneal dialysis solution. In some embodiments, the dose of metabolic enzyme can be about 0.5, about 1.0, about 1.5, or about 2.0 g per 100 ml of peritoneal dialysis solution. In some embodiments, the dose can be about 1.5 g of enzyme per 100 ml of peritoneal dialysis solution. In some embodiments, a mammal, eg, a human, can be administered about 10 ml, 20 ml, 30 ml, 40 ml, 50 ml, or 60 ml peritoneal dialysis solution per kg body weight per cycle. The duration of each cycle of peritoneal dialysis can range from about 2 to about 12 hours, such as from about 4 to about 12 hours, or from about 2 to about 8 hours. In some embodiments, the duration of each cycle of peritoneal dialysis can range from about 4 to about 6 hours. Multiple cycles, such as peritoneal dialysis for up to several weeks or months can be performed.

  Measure the concentration of metabolites, such as oxalate, in mammals in biological samples such as blood, serum, plasma, urine, or body fluids including peritoneal fluid before, during and after peritoneal dialysis be able to. Metabolite concentrations can also be measured with a peritoneal dialysis solution in the circulation. In some embodiments, the metabolite concentration is determined from other biological samples, such as samples taken from tissues, such as the eyeball, bone marrow, kidney, liver, or heart, or stomach, proximal small intestine, distal small intestine, cecum. It can be measured on samples taken from one or more of the contents of the colon, rectum, or rectum. Mammalian metabolites, such as oxalate concentrations, can be reduced by the methods described herein, such as the method of peritoneal dialysis described herein.

Animal models Animal models of hyperoxalicemia and / or peritoneal dialysis are also provided. In some embodiments, the non-human animal model can include a mammal. In some embodiments, the mammal can be a pig. Animals can be administered with continuous infusion of oxalate. Administration can be carried out using methods known in the art. For example, a first device can be inserted into an animal to administer oxalate with continuous infusion. In some embodiments, the first instrument can be selected from a catheter or pleural trocar. The catheter can be inserted percutaneously with or without surgery. In some embodiments, the catheter can be a peripheral venous catheter, such as VENFLON ™ or CATHLON ™. In some embodiments, an infusion pump can also be used. The oxalate concentration of the solution to be administered can be about 0.1% to about 10% (w / w). In some embodiments, the oxalate concentration can be 1%. For example, the solution to be administered can contain 1% sodium oxalate and 0.9% NaCl (saline). The rate, volume, and duration of infusion can be selected based on the desired blood oxalate concentration in the animal. For example, the rate of infusion can be from about 0.05 to about 2 mL / min. In some embodiments, the rate of infusion is about 0.15 to about 0.40 mL / min, such as about 0.17 mL / min, 0.20 mL / min, 0.27 mL / min, or 0.35 mL / min. Can be minutes. The volume of infusion can be about 1 to about 8 mL / kg body weight. In some embodiments, the volume of infusion can be about 3 to about 5 mL / kg body weight, for example about 4 mL / kg body weight. The duration of the infusion can be about 1 to about 72 hours. In some embodiments, the duration of the infusion can be about 4 to about 6 hours, such as 5 hours. In some embodiments, the infusion can be performed for up to several weeks. In some embodiments, bolus injection can be performed without a pump. For example, bolus injection can be performed at time intervals. In some embodiments, the time interval can be every 10, 20, 30, 40, 50, or 60 minutes.

  Before, during and after infusion or peritoneal dialysis, the oxalate concentration of the animal model can be measured in biological samples such as body fluids including blood, serum, plasma, urine, or peritoneal fluid. Oxalate concentration can also be measured with a peritoneal dialysis solution. In some embodiments, the oxalate concentration is determined from other biological samples, such as samples taken from tissues, such as the eyeball, bone marrow, kidney, liver, or heart, or stomach, proximal small intestine, distal small intestine, cecum, Measurements can be made on samples taken from one or more of the colon or rectal contents. The method described herein, for example, the method of peritoneal dialysis described herein, can reduce the oxalate concentration in an animal.

  The following examples provide exemplary embodiments of this disclosure. Those skilled in the art will recognize that many modifications and changes can be made without changing the spirit or scope of this disclosure. Such modifications and changes are encompassed within the scope of this disclosure. The examples do not limit this disclosure in any way. The contents of all patent and non-patent documents described in this publication are incorporated by reference in their entirety.

The examples described below are intended to be merely illustrative of the invention and are not intended to limit the invention in any way. The examples are not intended to represent that the following experiments are all or unique of the experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (eg amounts, temperature, etc.) but some responsibility for experimental error and deviation must be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

Example 1
The study was carried out on pigs (n = 4-6), each weighing approximately 20 ± 2 kg. The animals were maintained on a 12 hour day-night cycle with a light period of 6.00-18.00 (6 am-6pm) and a dark period of 18.00-06.00 (6 pm-6am). Pigs were individually housed in a bowl equipped with a dry sample bowl, a drinking nipple and a continuous heating lamp (150 W). Pigs were able to move freely within the cage and were visible to each other when not experimenting.

  The pigs were fasted overnight and then pre-administered with azaperone (Strensil, Janssen Pharmaceutical, Belgium, 4.0 mg / kg IM) prior to transport and further handling. In surgery, the pig is anesthetized with isoflurane mixed with 2% air (Forene 100%, Abbott Scandinavia AB, Solna, Sweden), and the closed circulatory system (Komesaloff Medical Developments, Melbourne Medical, Australia) is used. Supplemented (0.5 L / min).

  Two catheters (Standards Silicone Tubing, Helix Medical Carpinteria, CA, USA) were inserted into the right external jugular vein of the pig. One catheter for blood collection was inserted at a depth of 6 cm and another catheter for continuous infusion of sodium oxalate was inserted at a depth of 8 cm, both of which were 30 cm long (I. D. 1.02 mm and OD 2.16 mm). A catheter was inserted through a 4 cm long incision in the neck between the tracheal junction and the mandibular axis.

  A health record was recorded for each animal from the start of the study. Health records were the criteria for the next exclusion. Twice a day (2% of body weight per powdered feed) all young growing pigs (53908 VAXILL 320 P BK, Lantmannen, Sweden) were fed cereal feed. This amount is equivalent to the amount of food consumed when given without limitation in approximately the same state.

  To perform peritoneal dialysis (PD), 1 cm wide skin and myotomy, the peritoneal cavity, the end within 30 cm (id 3.8 mm and od 4.4 mm) of the tube length and a length of 2-3 holes A 30 cm tube was inserted. To close the “hole” in the skin, a grommet incision was sutured to the surrounding tissue for further catheter stabilization. Within 5 minutes, a PD solution based on pig body weight (20-40 mL / kg) was injected and the solute was drained using the same tube.

Example 2
Porcine model of hyperoxalate and establishment of PD 0.20 or 0.35 mL / min with an infusion pump (KD Scientific Syringe Pump Legato 100, KD Scientific Inc. Holliston, Ma, USA) with isoflurane to all perceptive hemp Continuously through the jugular vein at a constant rate of 1% sodium oxalate, Sigma-Aldrich Chemicals in 0.9% NaCl (saline); or the amount of oxalate is the total food intake per day Sodium oxalate was injected as 1% (w / w), for example, 10 g oxalate per kg total food intake per day. Injections were performed at a rate of 0.20 or 0.35 mL / min during 4-6 hours of injection such that the titration concentration was plasma oxalate concentration of 100-200 μmol / L. At the same time, PD was started as an oxalate injection with a PD volume of 40 mL / kg of pig body weight. A 20 kg pig having a blood volume of 2 L was administered with 800 mL of PD solution (GAMBROSOL TRIO ™ 10, glucose H, 3.9%, pH 6.3). The pH of PD was 7.4 5 to 10 minutes after the start of PD. As shown in FIGS. 1a and 1b, at rates of injection of 0.2 mL / min and 0.35 mL / min, hyperoxalateemia at about 60-100 μmol / L and about 120-180 μmol / L for 4 hours, respectively. Came. The average infusion time was about 4 hours.

  Blood samples were taken at 30 minutes between infusion and peritoneal dialysis and just before the start of infusion for pretreatment measurements. Each blood sample was quickly transferred to a tube for plasma and / or serum collection and kept on ice until centrifuged at 4 ° C. Further, samples collected at -20 ° C were stored until analysis of oxalate measurements using ion chromatography. Serum samples were diluted to 1/10 with Millipore water and filtered using a centrifugal filter (VWR International) with a molecular weight cut off (MWCO) of 3 kDa. 0.5 mL of the filtrate was transferred to a PP vial and placed in an autosampler for analysis on ICS-900 (Thermo Scientific).

  GAMBROSOL TRIO ™ 10 (Gambro AB, Lund, Sweden) was used as the dialysate for PD. Before infusion into the peritoneal cavity already filled with PD (40 mL / kg body weight), 1.5 g (65 unit dose) of powder OXDC (Oxalate decarboxylase from Bacillus subtilis, spray dried crystallin in 100 mL PD , Lot: 44 # 3, activity 10.9 u / mg total dry weight at optimum pH 4). The negative control was injected with GAMBROSOL TRIO ™ 10 only.

  PD samples were taken at the same time as blood samples every 30 minutes. Samples collected at −20 ° C. were stored until further analysis. The PD sample was diluted to 1/10 with Millipore water and filtered using a centrifugal filter (VWR International) with a molecular weight cut off (MWCO) of 3 kDa. 0.5 mL of the filtrate was transferred to a PP vial and placed in an autosampler for analysis using ion chromatography (ICS-900). At the end of the experiment, the volume of dialysate collected and drained from the peritoneal cavity was recorded.

  The pig was reused several times. At the end of the final experiment, pigs were slaughtered by overdose of pentobarbital (Allfatal vet. Omnidea AB, Sweden). Sap samples from the stomach, proximal small intestine, distal small intestine, cecum, colon and rectum were collected and the volume recorded. Samples were maintained at -20 ° C until analyzed for oxalate content.

  The juice sample was weighed to determine the oxalate content, then mixed with 9 volumes of 240 mM HCl and incubated for 1 hour at 60 ° C. to ensure complete dissolution of the oxalate crystals. Thereafter, the PD sample was diluted 1/100 with 0.4 M boric acid, and centrifuged at 14000 g for 30 minutes using a centrifugal filter (VWR International) with a molecular weight cut off (MWCO) of 3 kDa. 0.5 mL of the filtrate was transferred to a PP vial and placed in an autosampler of ion chromatography (ICS-900) for oxalate measurement. Samples were maintained at -20 ° C until analyzed for oxalate content.

The oxalate concentration in the sample was analyzed using ICS-900 with AS-DV autosampler (both Thermo Scientific) using ion chromatography (IC) method. ICS-900 is an IonPack AG4A-SC (2x50 mm) guard column, IonPack AS4A-SC (2x250 mm) analytical column, Displacement Chemical Regeneration Mode (DCR) Anion Micro Membrane SuMSL300 (all samples) From Thermo Scientific). The mobile phase was 1.8 mM Na ₂ CO ₃ /1.7 mM NaHCO ₃ at a flow rate of 0.5 mL / min.

The regenerant for AMMS 300 was 75 mN H ₂ SO ₄ . Statistical analysis was performed using Student's t-test or ANOVA or Prism 6 graph. Differences were considered significant when p <0.05.

  The pigs were infused with a constant rate of sodium oxalate (0.17 mL / min, 1% sodium oxalate in 0.9% sodium chloride) for 6 hours. Before PD dialysis, GAMBROSOL TRIO ™ 10 and powdered crystalline OXDC 1.5 g were mixed. Simultaneously with the start of oxalate infusion, PD + 1.5 g OXDC (about 65 U) was started at 40 mL / kg body weight (about 2 L blood / 800 mL PD).

  Peritoneal dialysis with OXDC started simultaneously with oxalate infusion was able to remove an average of about 20-40% of total serum oxalate from the circulation. As with human results with conventional PD, the removal rate was dependent on the plasma concentration of oxalate and the duration of dialysis. For example, Illies et al. , Kidney Int. 70: 1642-48 (2006)). The results of this experiment (FIG. 2 and Table 1) showed that OXDC decomposed oxalate in body fluids such as serum and PD from hyperoxalic acid pigs. The oxalate concentration in these body fluids was dependent on OXDC activity.

Example 3
In Vitro PD System Development Samples from the in vitro tests shown in FIGS. 3 and 5 were taken and stored at −20 ° C. prior to further analysis for oxalate measurement using the IC method described above. Statistical analysis was performed using Student's t-test or ANOVA or Prism 6 graph. Differences were considered significant when p <0.05.

  As shown in FIG. 3, dialysis was performed using a beaker containing 50 mM TrisCL pH 7.4, which closely resembled the blood compartment, and a dialysis bag, which resembled the peritoneal cavity filled with dialysis solution. Oxalate with a concentration of 100 μmol / L was added. Dialysis bags (10 kDa pore, Fisher) were used for GAMBROSOL TRIO ™ 10 (Gambro, Renal Products, Lund. Sweden), glucose High ± 1.5 g crystalline OXDC (about 15 mg / mL, or about pH 7.4, about 64 total). Unit dose OXDC) (TrisCL: GAMBROSOL TRIO ™ 10 ratio = 2.5: 1, approximately the same as the ratio of dialysis performed in humans). The bag pH after 7.0 minutes of dialysis was 7.0. Samples were taken from bags and beakers for oxalate measurement at 0 minutes, 30 minutes, 60 minutes, 240 minutes and day and night (FIG. 4).

  As shown in FIG. 5, a beaker (similar to serum from a hyperoxalic acid pig) and GAMBROSOL TRIO ™ 10 (similar to serum from a pig with hyperoxalicemia) with a final concentration of 100 μmol / L sodium oxalate added. Gambro, Renal Products, Lund. Sweden, Glucose H ± 70 mg of crystalline OXDC (approximately 30 mg / mL, or OXDC with a total dose of approximately 3 units at pH 7.4) (serum: GAMBROSOL TRIO ™ 10 ratio = 2 Dialysis was performed using a dialysis bag (10 kDa hole, Fisher) containing 5: 1, approximately the same ratio of dialysis performed in humans. Samples were taken from bags and beakers for oxalate measurement at 0, 30, 60, 240 minutes and at night. The results shown in FIG. 6 show that the oxalate concentration in the bag with GAMBROSOL TRIO ™ 10 ± OXDC (representing “peritoneal dialysate”) decreased to approximately 17.4% of total basal serum oxalate after 1 hour of dialysis. And stayed within that range until the end of dialysis at 6 hours.

  To reveal the minimum dose of OXDC that can significantly reduce serum oxalate concentrations, various concentrations in GAMBROSOL TRIO ™ 10 at 37 ° C for up to 6 hours at a constant shaking speed of 130 rpm. In vitro test as described above with OXDC (approximately 30, 20, 15, 10, 5 and 1 mg / mL) and oxalate serum starting concentration of 100 μmol / L (serum with sodium oxalate as described above) Carried out. Samples were taken from the beakers at 0, 30, 60, 240 and 360 minutes for oxalate measurement.

  The results shown in FIG. 7 showed that serum oxalate concentration was reduced by 35% after 6 hours incubation with 10 mg / mL OXDC. As a result, OXDC used in the experiment at pH 7.4 (serum and body fluid pH) had an efficacy of less than 0.1 unit dose / mg substrate.

Claims (19)

A composition comprising a peritoneal dialysis solution and at least one metabolic enzyme or functional fragment thereof, wherein the at least one metabolic enzyme or functional fragment thereof comprises an oxalate decal Boki Schiller peptidase, composition.   The at least one metabolic enzyme or functional fragment thereof further comprises an additional metabolic enzyme or functional fragment thereof selected from oxalate degrading enzyme, urate degrading enzyme, ureolytic enzyme, bilirubin degrading enzyme, and phenylalanine degrading enzyme; The composition of claim 1. (A) the oxalate degrading enzyme is selected from oxalate oxidase and oxalyl-CoA decarboxylase;
(B) the urate degrading enzyme is urate oxidase (uricase);
(C) the ureolytic enzyme is urease;
(D) the bilirubin degrading enzyme is bilirubin oxidase, and (e) the phenylalanine degrading enzyme is phenylalanine hydroxylase or phenylalanine ammonia lyase,
The composition according to claim 2.   The composition according to any one of claims 1 to 3, wherein the at least one metabolic enzyme is covalently modified.   5. The composition of claim 4, wherein the at least one metabolic enzyme is PEGylated. 6. A composition according to claim 2 or 3, or when quoting claim 2 or 3, wherein the additional metabolic enzyme is soluble or crystalline.   6. A composition according to any preceding claim, wherein the at least one metabolic enzyme is cross-linked or not cross-linked.   The composition according to any of claims 1 to 7, wherein the peritoneal dialysis solution comprises at least one osmotic agent, at least one electrolyte, and at least one organic acid salt. The composition is
(A) can reduce the metabolite concentration of a mammal, and / or (b) urine, blood, plasma, serum, peritoneal fluid, eyeball, bone patch, kidney, liver, heart, and stomach, proximal Reduce the metabolite concentration of a biological sample selected from the contents of the small intestine, distal small intestine, cecum, colon, and rectum;
The composition according to any one of claims 1 to 8.   10. The composition of claim 9, wherein the metabolite is selected from oxalate, uric acid, urea, bilirubin, and phenylalanine.   11. A composition according to any of claims 1 to 10 for use in a method of reducing mammalian metabolite concentration.   12. The composition for use according to claim 11, wherein the method further comprises detecting the metabolite concentration of the mammalian biological sample.   13. A composition for use according to claim 12, wherein the biological sample is selected from urine, blood, plasma, serum, and peritoneal fluid or taken from tissue.   The method reduces the metabolite concentration by at least 10%, and optionally the decrease is urine, blood, plasma, serum, and peritoneal fluid, eyeball, bone follower, kidney, liver, heart, and stomach, proximal small intestine. 14. A composition for use according to any of claims 11 to 13, measured in a biological sample selected from the contents of, distal small intestine, cecum, colon, and rectum.   11. A composition according to any preceding claim for use in a method of treating a disorder associated with increased mammalian metabolite concentration. Hyperoxalicemia secondary to the disease selected from: (a) oxalic acid disease, hyperoxalicemia, or kidney injury, bone disorder, liver disorder, gastrointestinal disorder, and pancreatic disorder Selected from
(B) Oxalosis, hyperoxaluemia, primary hyperoxaluria, intestinal hyperoxaluria, idiopathic hyperoxaluria, and end-stage renal injury, ethylene glycol poisoning, cystic pulmonary fibrosis, inflammatory Selected from secondary hyperoxalicemia for diseases selected from bowel disease, urolithiasis, nephrolithia, and chronic kidney disease, or (c) hyperuricemia, gout, uremia Selected from symptom, hyperbilirubinemia, jaundice, hyperphenylalaninemia, and phenorketonuria (PKU),
A composition for use according to claim 15.   17. The method of any of claims 11 to 16, wherein the method comprises adding the at least one metabolic enzyme or functional fragment thereof to a peritoneal dialysis solution and administering the added peritoneal dialysis solution to the mammal. A composition for use according to claim 1.   18. A composition for use according to any of claims 11 to 17, wherein the metabolite is selected from oxalate, uric acid, urea, bilirubin, and phenylalanine.   19. A composition for use according to any of claims 11 to 18, wherein the method is combined with hemodialysis.